title: Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.
abstract: Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus.
authors.affiliation: Department of Biological Sciences, Brock University, St Catharines, ON, L2S 3A1, Canada.
authors.forename: Vonny
authors.initials: V
authors.lastname: Salim
authors.forename: Fang
authors.initials: F
authors.lastname: Yu
authors.forename: Joaquín
authors.initials: J
authors.lastname: Altarejos
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Luca
mesh_terms: D030008:Catharanthus / Q000201:enzymology / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D039823:Iridoids / Q000378:metabolism; D055442:Metabolome; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D046948:Secologanin Tryptamine Alkaloids / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C432876:7-deoxyloganic acid; D039823:Iridoids; D010940:Plant Proteins; D046948:Secologanin Tryptamine Alkaloids; D006899:Mixed Function Oxygenases
keywords: 7-deoxyloganic acid hydroxylase; bioinformatics-guided candidate gene identification; cytochrome P450; internal phloem parenchyma cells; spatial separation of secologanin biosynthesis; virus-induced gene silencing in Catharanthus roseus